The present invention relates to a method for producing carrier-bound enzymes, in which a carrier which has functional groups capable of forming covalent bonds is coated with an enzyme.
When carrying out reactions catalyzed by enzymes, there is a choice of basic alternatives between homogeneously catalyzed methods using free enzymes and heterogeneously catalyzed methods using carrier-bound enzymes. Compared to free enzymes, carrier-bound enzymes have many advantages, among which their easier reusability and their outstanding suitability for processes which have to be carried out continuously deserve particular mention. Both carriers made of organic and carriers made of inorganic materials may be considered for use as carriers. Inorganic carriers have the advantage of low compressibility, particularly in industrial-scale, continuous processes in which the carrier-bound enzymes are used in column-shaped reactors with enzyme beds of appropriate height. But whereas organic carriers often naturally have functional groups to which the enzymes can be bound, inorganic carriers as a rule do not have such functional groups. Inorganic carriers must therefore be provided with such functional groups in a separate step. Currently, treatment with silanes carrying inorganic functional groups and organic functional groups has found general acceptance as the means of choice for this purpose. The classical representative of such silanes is gamma-aminopropyltriethoxy silane, in which an Si--O--bond to the carrier is produced through hydrolysis of the ethoxy groups, while the aminopropyl group remains as an organic functional group for forming bonds to the enzyme.
For example, U.S. Pat. No. 3,519,538, as the basic literature reference for a method of the aforementioned type, proposes to treat inorganic carriers with silanes of the type mentioned above and then to bond the enzymes to the thus functionalized carrier, referred to therein as an aminoalkyl silane derivative, either directly or by inserting a "spacer". Non-siliceous metal oxides such as aluminum oxide, hydroxyapatite or nickel oxide, and siliceous substances such as porous glass, silica, wollastonite, silica gel or bentonite are listed therein as suitable inorganic carriers.
While U.S. Pat. No. 3,519,538, having disclosed the aforementioned process, is credited with showing that inorganic carriers can be used to produce carrier-bound enzymes and how they can be so used, U.S. Pat. No. 4,230,803 develops the aforedescribed method further, in that this document teaches how the particular carrier which is optimum with regard to its pore size and pore distribution, can be determined from among several carriers under consideration, and how the concentration of the enzyme solution, with which the functionalized carrier is to be reacted, can be determined in order to obtain a maximally active preparation using a minimum amount of enzyme, the preparation also being distinguished in particular by the fact that the activity of the carrier-bound enzyme is the same or only slightly less than the activity of the enzyme in the free state.
Although excellent carrier-bound enzymes with high initial activity can be produced according to the method of U.S. Pat. No. 4,230,803, these and essentially all other carrier-bound enzymes suffer, in principle, from only limited stability, i.e. the activity of the carrier-bound enzyme decreases at a greater or lesser rate under the conditions of practical use.
U.S. Pat. No. 4,533,633 counters this drawback with a carrier-bound enzyme produced based on SiO.sub.2 carriers, by contacting the substrate with shaped bodies of SiO.sub.2 or alumosilicate before the reaction at the carrier-bound enzyme. Alternatively, with a type of carrier-bound enzyme which is similar as far as the carrier is concerned, U.S. Pat. No. 4,665,025 proposes to add water-soluble silicate to the substrate. However, these two last-mentioned improvements are restricted to carrier-bound enzymes with carriers which consist entirely or at least substantially of siliceous material.
The use of glutaric dialdehyde and polyethylene imine in producing carrier-bound enzymes is already known. For example, U.S. Pat. No. 4,141,857 describes the treatment of porous inorganic carriers with polyethylene imine and glutaric dialdehyde in order to produce, as stated therein, combined organic/inorganic base material from an inorganic porous carrier with an organic polymer material adsorbed and entrapped in its pores. The enzyme is then bound to the organic polymer material in a subsequent step.
The treatment of the carrier with polyethylene imine and glutaric dialdehyde thus corresponds to the previously described silanizing in that a virtual intermediate layer is produced between the carrier and the enzyme, which layer is capable on the one hand of forming a bond to the carrier--by siloxane formation in the case of silylation and by positive attachment of the polymer produced in the pores of the carrier in the case of treatment with polyethylene imine and glutaric dialdehyde--and on the other hand forming a bond to the enzyme. In other words, the "combined organic-inorganic basic material" obtained through treating porous inorganic carriers with polyethylene imine and glutaric dialdehyde corresponds to the "carriers which have functional groups capable of forming covalent bonds" defined in the general process described in the background section above.
What has been said about U.S. Pat. No. 4,141,857 also applies to U.S. Pat. No. 4,438,196 in which activated carbon is treated with polyethylene imine and glutaric dialdehyde in order to produce a product which corresponds to the combined organic/inorganic base material of U.S. Pat. No. 4,141,857.
Even more remote are processes using polyethylene imine, represented for example by European Patent No. 133,531, in which polyethylene imine, optionally in combination with other substances, is used to flocculate enzymes dissolved in water or microorganism cells suspended in water and containing intracellular enzymes. The flocculated material is then processed in further steps to form immobilized biocatalysts.